Considerably more information has been acquired about the three myosin isoenzymes of Acanthamoeba. A 16-amino acid sequence has been identified which contains the ATP-binding site of myosin II: Glu-Ser-Gly-Ala-Gly-Lys-Thr-Gln-Asn-Thr-Me2Lys-Lys-Val-Ile-Gln-Tyr. Radioactive nucleotide is photoaffinity crosslinked to the Gln at position 8 in this sequence. The sequence is highly homologous to a sequence in nematode and rabbit striated myosins that lies in the previously identified "ATP-binding" region of those myosins but differs from the two sequences that are photoaffinity labeled in rabbit myosin by two different analogues of ATP. A 68-kDa head fragment has been purified from tryptic digests of myosin II and shown to have complete Ca-ATPase activity but no actin-activated MgATPase activity. It did not bind detectably to F-actin. When covalently crosslinked to F-actin by EDC, the 68-kDa showed very high actin-activated ATPase activity; suggesting that the low activity of the non-crosslinked head was due to its poor binding. It is likely that phosphorylation regulates the activity of intact, native myosin II in the same way, i.e. by affecting its binding to F-actin. Kinetic studies of the ATPase activity of myosin IA and IB as a function of myosin concentration at fixed actin concentration have led to the hypothesis that myosin I heavy chain contains two actin-binding sites. This hypothesis was supported by evidence that single molecules of myosin I crosslink F-actin (as measured by the increase in low-shear viscosity) and, in the presence of ATP and when the myosin I is phosphorylated, superprecipitate F-actin. Thus, monomolecular, non-filamentous myosin I can support contractile activity which means that the heavy chain of 125-130 kDa contains all that is required for contractile activity and that myosin filaments are not needed.